A metastasis‐associated microRNA‐based liquid biopsy signature for risk‐stratification in colorectal cancer: a multicenter cohort study

Dear Editor, Colorectal cancer (CRC) continuously sheds various subcellular components into the bloodstream, including microRNAs (miRNAs).1–3 In the current study, we systematically and comprehensively profiled miRNAs in patients with CRC to test the hypothesis that miRNAs highly expressed in metastases are shed into the bloodstream and can act as promising blood-based biomarkers to aid in clinical decision-making, which led us to identify clinically translatable circulating miRNAs for recurrence prediction in patients with stage II and III CRC. The overall study design is illustrated in Figure S1. During the discovery phase, we analysed the GSE54088 dataset4 to identify miRNAs differentially expressed in liver metastases compared to normal liver as well as normal colorectal mucosa. The heatmaps depicting the expression of seven candidate miRNAs are presented in Figure 1A, which clearly highlight that these candidates are unique and likely represent liver metastasis. Next, we obtained 25 formalin-fixed, paraffin-embedded metastatic CRC tissues (from the liver or lung), 22 matching normal liver or lung tissues, and 6 matching normal colorectal mucosae to examine the differential expression of the candidate miRNAs using reverse transcription polymerase chain reaction (qRT-PCR). It was quite re-assuring to witness that all sevenmiRNAs were upregulated in liver or lung metastases tissues (Figure S2A,B). In order to evaluate the clinical utility of candidate miRNAs as blood-based biomarkers, we analysed blood specimens collected from two independent clinical cohorts before and after primary CRC resection. As a result, we identified threemiRNAs (miR-210, miR-425* andmiR-141) that were highly expressed in both preand post-operative blood specimens from CRC patients with distant metas-

A metastasis-associated microRNA-based liquid biopsy signature for risk-stratification in colorectal cancer: a multicenter cohort study Dear Editor, Colorectal cancer (CRC) continuously sheds various subcellular components into the bloodstream, including microRNAs (miRNAs). [1][2][3] In the current study, we systematically and comprehensively profiled miRNAs in patients with CRC to test the hypothesis that miRNAs highly expressed in metastases are shed into the bloodstream and can act as promising blood-based biomarkers to aid in clinical decision-making, which led us to identify clinically translatable circulating miRNAs for recurrence prediction in patients with stage II and III CRC.
The overall study design is illustrated in Figure S1. During the discovery phase, we analysed the GSE54088 dataset 4 to identify miRNAs differentially expressed in liver metastases compared to normal liver as well as normal colorectal mucosa. The heatmaps depicting the expression of seven candidate miRNAs are presented in Figure 1A, which clearly highlight that these candidates are unique and likely represent liver metastasis.
Next, we obtained 25 formalin-fixed, paraffin-embedded metastatic CRC tissues (from the liver or lung), 22 matching normal liver or lung tissues, and 6 matching normal colorectal mucosae to examine the differential expression of the candidate miRNAs using reverse transcription polymerase chain reaction (qRT-PCR). It was quite re-assuring to witness that all seven miRNAs were upregulated in liver or lung metastases tissues ( Figure S2A,B).
In order to evaluate the clinical utility of candidate miRNAs as blood-based biomarkers, we analysed blood specimens collected from two independent clinical cohorts before and after primary CRC resection. As a result, we identified three miRNAs (miR-210, miR-425* and miR-141) that were highly expressed in both pre-and post-operative blood specimens from CRC patients with distant metas-tasis (stage IV) compared to patients without distant metastasis (stages I-III) in both cohorts (cohort 1 [N = 136]: Figure 1B, Figure S3A, cohort 2 [N = 180]: Figure 1C, Figure  S3B). The high-expression of these three miRNAs in stage IV CRC indicates that these tumour-derived markers are likely shed into systemic circulation, making them attractive candidates for development as liquid-biopsy markers for the identification of metastasis in patients with CRC. We analysed the associations between the expression levels of these miRNAs and key clinicopathological characteristics (Table S1: cohort 1, Table S2: cohort 2); indicating that our candidates are more related to haematogenous metastasis instead of lymphatic metastasis in CRC patients. In other words, we speculate that these miRNAs might be shed from CRC cells through venous invasion and could be detectable in blood.
To further investigate whether the circulating metastasis-associated miRNAs could be used to predict prognosis in patients with CRC, we next analysed the OS of all CRC patients in our cohorts (stages I-IV) and the disease-free survival (DFS) of the patients with stages II and III CRC. Kaplan-Meier analyses revealed that patients with high preoperative or postoperative miR-210 and miR-425*, or high postoperative miR-141 levels exhibited significantly worse OS in both clinical cohorts ( Figure S4A: cohort 1, Figure S4B: cohort 2). Kaplan-Meier analyses revealed that patients with high preoperative miR-210 or postoperative miR-425* levels had significantly worse DFS in both cohorts ( Figure 2A: cohort 1, Figure 2D: cohort 2). We performed univariate Cox proportional hazards model analyses for predicting the DFS in the cohort 1 and 2 ( Figure 2B Next, we constructed DFS prediction models for patients with stage II and III CRC in the cohort 2, by evaluating the predictive potential of the combination of circulating preoperative miR-210 and postoperative miR-425* using Cox proportional hazard models. This combination yielded an area under the time-dependent receiver operating characteristic (ROC) curve (AUC) of 0.795 ( Figure 3A). This model could also robustly predict DFS (HR: 9.10, 95% CI 2.47-33.48; P < .001). Thereafter, we constructed a new DFS prediction model combining the two-circulating metastasis-associated miRNAs with these two clinicopathological variables (T4 classification and venous invasion positivity), which yielded an AUC of 0.859 ( Figure 3A). This model predicted DFS even more efficiently (HR: 12.02, 95% CI: 3.59-40.22; P < .001; Figure 3B). In addition, we evaluated this DFS prediction model in patients with stage II and III CRC separately, demonstrating that high scores were associated with lower DFS rates for patients in both groups (P < .001 for both; Figure 3C,D). Collectively, these results demonstrate the clinical significance of our circulating miRNA combination for risk stratification in patients with stage II and III CRC, as well as stage II CRC for whom actionable biomarkers are urgently needed in the clinic for the identification of optimal subgroup of patients who can benefit from adjuvant chemotherapy.
Several investigators have identified and demonstrated the clinical significance of metastasis-specific tissue-based biomarkers that were upregulated in metastases compared to primary CRC. 5,6 However, high reliance on such comparisons might have led to missing significant candidate miRNA biomarkers that are upregulated in both metastasis and primary CRC compared to normal tissue. Therefore, in the in silico miRNA discovery phase of this study, we aimed to identify candidate miRNAs that were upregulated in liver metastasis compared to normal colon mucosa and surrounding normal liver, ensuring that we can successfully develop clinically actionable circulating metastasis-associated miRNAs.
Our study has potential limitations, given its retrospective nature. We did not have access to the status of microsatellite instability, which might be relevant additional clinical information for treatment selection for adjuvant therapy. Therefore, our results must be validated in a prospective, multi-centre clinical trial, to evaluate the potential of this circulating miRNA combination for recurrence prediction.
In conclusion, we provide novel and clinically important evidence that our circulating metastasis-associated miRNA signature which can be detected in pre-operative and early post-operative blood samples, can effectively risk-stratify patients with stage II and III CRC. These circulating miRNAs offer tremendous clinical potential for directing personalised treatment regimens and the clinical management of patients with this lethal malignancy.

C O N F L I C T O F I N T E R E S T
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.